It is well known that antigen-polyacrylamide gels can be used to entrap antibodies. It is also well known that certain enzymes, e.g., trypsin, amylase and ribonuclease have been entrapped and used as attractive antigens in such gels (Science 142:678-679, 1963). Antigens can also be purified in a similar manner using specific antibodies in the acrylamide gels to attract the antigens (Journal of Histochemistry and Cytochemistry, 23(2):146-148, 1975). These tendencies for certain antibodies and antigens to attract one another can be put to practical use. For example, an affinity chromatography procedure using a venom-polyacrylamide gel has been used to further purify equine source antivenin (Proc. West. Pharmacol. Soc. 25:185-192, 1982). That is to say, venom-polyacrylamide gels have been used to further purify a commercially available antivenin, Antivenin (Crotalidae) Polyvalent (“ACP”-Wyeth Laboratories, Box 8299, Philadelphia, Pa., U.S.A.) which is normally purified by ammonium sulfate precipitation procedures (American Journal of Emergency Medicine, 1:83-93, 1983). It is also well known that various enzymes such as trypsin, chymotrypsin and papain can be used to split antibody molecules into fragments having higher activities and lower toxicities than the original antibody molecule (see for example, U.S. Pat. No. 4,012,502). Such procedures normally involve removing desired antibody containing protein fragments in a fractionating column, solution digesting these antibody containing protein fragments with various enzymes and then isolating the sought after antibody fragments in Sepharose columns.
Conceptually, a whole antibody molecule, commonly referred to as IgG, is often thought of as being comprised of three fragments connected in a Y shape. The two upper fragments are each referred to as F(ab) fragments. The stem of the Y is commonly referred to as the F(c) fragment. Each of the F(ab) fragments can be split from each other and from the F(c) fragment. The whole antibody, IgG molecule can also be cleaved into a larger fragment commonly referred to as a F(ab)2 fragment and a F(c) fragment. This cleavage takes place in such a manner that an F(ab)2 fragment is comprised of two attached F(ab) fragments. It is well known in the art that exposure of IgG molecules to papain produces F(ab) fragments and that pepsin digestion produces F(ab)2 fragments. It is also well known in the art that the smaller F(ab) fragments are less likely to cause undesired immunogenic reactions. A general rule is that, given possession of the antibody active site, the smaller the antibody molecule the better.
IgG, F(ab) fragments and F(ab)2 fragments often have separate utilities. Furthermore, F(ab) and F(ab)2 fragments may sometimes be utilized together. Hence processes for obtaining antibodies in each of these forms are highly desirable. For example, F(ab) fragments have the same affinity for antigens as IgG molecules, but they have lower molecular weights. Consequently, for reasons hereinafter more fully discussed, they can be more quickly distributed in the body and then filtered and excreted by the kidney. IgG molecules on the other hand are generally too large to be excreted by kidney functions. This means that whole IgG cannot distribute to tissue sites and neutralize toxins similar to F(ab) nor can the IgG and toxin combination be excreted by kidneys. However, IgG molecules can perform other useful functions. The F(c) portion of their molecule normally acts as a signal marker for lymphocytes to recognize and phayocytize. This is the chief pharmacological reason for leaving an IgG molecule intact. If for example a sought after antigen molecule is already a large molecule, the use of F(ab) fragments is limited. Even though the F(ab) fragments can be used to neutralize large antigen molecules, the kidneys still will not be able to excrete them. In such cases it may be better to use a whole IgG molecule to find the large antigen molecule so that the entire molecule assembly is phagocytized. See for example, Clin, Exp. IMMUNOL, 36:384-396 (1979).
Another advantage to having the ability to break IgG antibodies into F(ab) or F(ab)2 fragments is found in the pharmacological concept of volume of distribution. Volume of distribution is that volume of the body in which a given drug is dissolved. Circulating blood has a certain volume, but the body's water volume is much greater. For most IgG proteins, the volume of distribution is limited to the volume of circulating blood. However, for smaller molecules such as F(ab) fragments, the volume of distribution may be the total volume of body water. Furthermore, smaller molecules such as F(ab) fragments often have the ability to cross certain physiological systems and barriers such as, for example, the blood/brain barrier. Larger antibodies do not have this capability. Consequently, neurotoxins may not be accessible to IgG molecules since the IgG molecules are confined to the circulating blood system. However many neurotoxins, including some snake venoms, may be accessible to F(ab) fragments because F(ab) fragments usually have a volume of distribution which includes water; hence the F(ab) fragments may be capable of crossing the blood/brain barrier in both the incoming and outgoing directions. In such cases F(ab) fragments may be used to excrete many kinds of neurotoxins which are not otherwise accessible to IgG antibodies. Thus from both the pharmacological and toxicological point of view, the ability to separate and purify a given antibody into selected fragments is of great significance to its application.
Antibody purity is a particularly important issue in the antivenin preparation art. Antivenin is a suspension of venom-neutralizing antibodies prepared from the serum of animals (typically horses) hyperimmunized against a specific venom or venoms. Horse and other animal serums are often digested with pepsin to obtain antivenin agents which are then precipitated out of the solution. Monovalent Bothrops (Laboratories “MYN”, S.A., Av. Coyoacan 1707, Mexico City 12, D.F., Mexico), Anticrotalic (Instituto Butantan, Ciaxa Postal 65, Sao Paulo, Brazil) and Centiviperin (Institute Pasteur d'Algerie, Rue Docteur Layeran, Alger, Algeria) are examples of antivenin antibodies which are pepsin digested and then precipitated with ammonium sulphate.
Unfortunately, such enzyme digestion and ammonium precipitation procedures do not remove all foreign proteins from horse serum derived antivenins. Consequently, some bite victims undergoing antivenin treatment suffer extreme life threatening allergic reactions to those foreign proteins which are not removed from the horse serum by prior art purification procedures. Less life threatening serum sickness reactions are also common. The exact mechanism for these allergic reactions has not been elucidated. For example they do not appear to be precisely related to previous exposure to horse antigen. Many researchers believe they may be due to anticomplementary activity of the serum. Another school of thought takes the position that the purity of the antivenin rather than the origin of the serum is the more critical factor in such allergic reactions. As a precaution however, all patients with known allergies to horses or horse serum are normally regarded as being at risk for serious anaphylactic reactions if given horse serum antivenin without adequate preparation. Therefore, any new and more efficacious procedures for purifying antibodies in general should be considered as being particularly important to the antivenin preparation art.